journal of regulatory science 02 (2015) 15–19 method

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Regulatory Science

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Journal of Regulatory Science 02 (2015) 15–19

RegulatoryScience

Method Validation for the Analysis of Multiple Weight Loss Drugs inDietary Supplement Materials by LC-MS/MS

Kevin Trana,∗, Douglas Monroea, Fenhong Songa, Aref El-Demerdasha

aKansas District Laboratory, Food and Drug Administration, 11510 W 80th St, Lenexa, KS, 66214

Abstract

A method validation is reported for the detection and quantification of multiple weight loss drugs in dietary supplement materials. The samplepreparation was followed as described in LIB 4549 and the mass spectral measurements were accomplished using liquid chromatography tandemmass spectrometry with electrospray ionization. A method validation was performed at various fortification levels (250 and 1000 µg/g) for all 11drug compounds. The overall recoveries with standard deviation averaged 93.8 ± 6.65% with the majority of individual recoveries in the rangeof 76% - 110%. The overall limits of quantification (LOQ) for analytes averaged at 10 ng/g. An overall indicator of linearity (R2) averaged at0.9993 (0.0 250.0 ng/mL). The precision of five replicates of the 50 ng/mL level averaged at 98.4± 0.41% with < 1%.

Keywords:weight loss drugs, sibutramine, LC-MS/MS

1. Introduction

Under the Dietary Supplement Health and Education Act(DSHEA) of 1994, a dietary supplement in the U.S. is definedas a product that is intended to supplement the diet which mayinclude vitamins, minerals, herbs or other botanical, amino acids,or concentrates, and extracts of these. However, prescriptiondrugs such as weight loss and other drugs are being detected indietary supplement products at the FDA and other labs. Theseillegal products can cause serious problems when consumerstake them without knowing the presence of drugs [1, 2, 3, 4].Furthermore, according to the National Institute of Technology(NIST) and National Institute of Health (NIH), approximately75% of the U.S population takes dietary supplements either bypills, juices or both representing an annual expenditure of morethan $20 billion [5]. There are many dietary supplement prod-ucts coming in US markets every year, promoted and availableon the internet. To prevent adulterated products and to meetwith the challenge of testing too many products, our labora-tory has developed a method to analyze dietary supplements formultiple weight loss drugs including sibutramine, which is oneof the most common adulterants in dietary supplements soldfor weight loss purposes [4]. Here we report our results of this

∗Corresponding author: Kevin Tran, Phone:913-752-2714.Email:[email protected],Kansas District Laboratory, Food and DrugAdministration.

method validation on dietary supplements provided by CFSAN.The results indicate our developed method is suitable for confir-mation and quantification of selected adulterated drugs includ-ing sibutramine and ten others in dietary supplements.

2. Experimental

Method validation was accomplished by evaluation of se-lectivity, precision, standard calibration linearity, standard cal-ibration accuracy, method detection limit and method limits ofquantification. The performance of the method validation wasevaluated using the method validation criteria outlined by FDA,Guidelines for the Validation of Chemical Methods for the FDAFoods Program. These criteria are listed in Table 1.

All mass spectral measurements were performed on an Agi-lent 6490 LC-MS/MS system with an electrospray source equippedwith an Agilent 1260 LC system. The LC-MS/MS data wereprocessed using Agilents Mass Hunter software.

2.1. Materials and ApparatusThe methanol and acetonitrile were HPLC high purity grade

(Burdick & Jackson, Morristown, NJ, USA). Ammonium for-mate was purchased from Fisher Scientific (Waltham, MA, USA).Milli-Q water was used throughout the validation (Millipore,18.2 Ω). Drug standards were obtained with high purity grade (98% pure) from Sigma Aldrich (St. Louis, MO, USA), TorontoResearch Chemicals (Toronto, Ontario, Canada) and from U.S.

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Pharmacopeia (Rockville, MD, USA). Individual stock solu-tions for the 11 drug compounds were prepared in acetonitrileat 1000µg/mL. The standard solutions were stored in a refrig-erator.

2.2. Calibration Standards

The injection of various calibration standards in acetoni-trile/ H2O (1:1) was used to construct the calibration curves(concentration versus peak areas). A series of dilutions weremade in methanol to prepare analytical standards containing 0,10, 20, 40, 80, 100 and 250 ng/mL concentrations.

2.3. Sample Preparation and Extraction

Five capsules or tablets were typically used to make samplecomposites. The tablet samples were ground into a fine pow-der to prepare a composite; capsule samples were emptied andmixed together. About 100 mg of composite was weighed for

each product and then extracted using 20 mL of acetonitrile in50 mL centrifuge tubes (30 x 115 mm polypropylene conicalFalcon tubes, Becton-Dickinson Labware). To determine thespike recoveries of the analytes, spiking standards were addedto portions of the sample composites before addition of the ex-traction solvent. The samples were shaken on a Burrell wrist-action shaker (Burrell Scientific, Pittsburgh, PA) for 30 minsthen centrifuged at 4000 rpm for 5 mins (Thermo ScientificMultifuge X3R). The supernatant was diluted for injection withacetonitrile/H2O (1:1). The samples and high-level-spike sam-ples were diluted 133 fold. On the other hand, low-level-spikesamples were diluted to 33 fold. All samples were then filteredusing 0.2 µm PTFE membranes in vials (GE Mini-Uni PrepPTFE Filter Media/Vials, 0.2 µm pore size). A 1 µL samplevolume was injected onto the HPLC-MS. For each sample

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matrix studied, a matrix blank, matrix spike and matrix spikeduplicate were prepared by weighing the same amount of sam-ple and performing the extraction procedures as described above.Each sample was fortified at two different concentrations (250and 1000 µg/g) prior to extraction.

2.4. Calculation

Sample results:amount f ound/capsule =

(mg. f ound)mg.composite x total.wt.o f .composite

number.o f .capsulewhere: mg found is the amount obtained from LCMS; mg

composite is the weight-out amount for extraction; total wt. ofcomposite is the total weight of all capsules used

Spike results:%spike.recovery =

mg. f ound−mg. f rom.capsulemg.std.added x100%

where: mg from capsule is defined as (mg composite x(amountfound/capsule)

Calculation for RPD:RPD =

spk1−spk2(spk1+spk2)/2 x100%

All statistical calculations were determined using MicrosoftOffice Excel 2010. These include calculations of average, stan-dard deviation (STDEV), relative standard deviation (RSD =

STDEV÷ Average x 100)

2.5. LC-MS/MS Determiantion

The LC-MS/MS system used for this validation consistedof Agilent 1260 LC interfaced to an Agilent 6490 LC-MS/MSwith an electrospray ionization source in the positive ionizationmode. The LC column was an Agilent 100 x 2.1 mm ZorbaxC18, 1.8 µm particle size. Gradient elution was performed us-ing 2 mM of ammonium formate in water with 0.05% formicacid as mobile A and 2 mM of ammonium formate in methanolwith 0.05% formic acid as mobile B. Gradient elution startedwith 80% B, increased to 95% B at 8.5 min and held for 4.5 min,the changed to 20% B at 13.5 min. The flow rate was 0.3 mLper minute and the runtime of the method was 15 min. The in-jection volume was 1 µL. Detailed LC-MS/MS conditions andparameters are listed in Table 2. The acquisition parameters foreach drug compound are listed in Table 3. The collision ener-gies were optimized with standard references.

3. Validation Results

3.1. Selectivity

Two official samples of dietary supplements received fromCFSAN were subjected for this validation. They representedsamples as real as consumers may purchase and intake. Theyare sold in the stores and all over internet as promising di-etary supplements for weight loss. Analysis selectivity usingLC-MS/MS is obtained by monitoring two or more multiplereaction monitoring (MRM) transitions, comparing and evalu-ating selected MRM response ratios, and by measurement ofthe chromatographic retention time (RT) of each analyte. Un-der conditions set for this validation, all peaks appeared wellwithin 5% of RT according to CVM Guidance for Industry [6].The ratio of three MS/MS transitions for sample and average ofstandards was also within 20% [6].

3.2. LinearityCalibration linearity was determined by evaluation of the

correlation coefficients, R2. Working standards in methanolwere prepared to construct the instrument calibration curves of0, 10, 20, 40, 80, 100, and 250 ng/mL. As shown in Table 4, theoverall R2 for all compounds was 0.9993 with the average of in-dividual R2 of each compound ranging from 0.9957 to 0.9999.

3.3. AccuracyInitial calibration (IC), continuing calibration verification

(CCV) and initial calibration verification (ICV) were preparedat 50 ng/mL. For spike recovery study, each sample matrix wasfortified at two different concentrations. Calculated amounts ofspike solutions were added prior to the extraction. Results ob-tained for all elements in this validation for analysis accuracywere within the successful ranges of criteria. The statistical datafor the overall recoveries are summarized in Table 5. The av-erage recoveries with standard deviation (STDEV) were from76.0 ± 7.40% to 110.6 ± 2.44%, respectively. The overall av-erage recovery and overall STDEV were 93.8 ± 6.65 % for allanalytes in these sample matrices. Desmethylsibutramine wasdetected in both matrix blanks. Thus, amounts of desmethyl-sibutramine recoveries reported in Table 5 are results after thededuction of found amounts. Finding of desmethylsibutraminewas reported in a package of sample analyses. Desmethylsibu-tramine is not a common adulterant but lately found in manydietary supplements [7]. On the other hand, sibutramine is thecommon adulterant in dietary supplements sold for weight loss[4]. It is an FDA-approved drug in 1997 used as an appetitesuppressant for weight loss and was withdrawn in 2010 due toassociated cardiovascular risks [1].

3.4. PrecisionThe within-batch precision was determined by analyzing

five replicate analyses of a solvent standard solution (50 ng/mL).The recoveries of five analyses were from 97.66% to 98.90%.The average recovery with STDEV was 98.4± 0.41% (RSD<1%).

3.5. Detection LimitsThe method detection limit (MDL or LOD) and limits of

quantification (LOQ) should be demonstrated in practice by ac-quiring signal/noise ratios>3 and acceptable recovery data atthe claimed LOQ. The LOQ was estimated at 10 ng/mL in thisvalidation. The estimated LOQ level in this study was in fact100+ times lower than the typical levels of concern for thesetypes of products. When added to dietary supplements, thesetypes of weight loss drugs are usually present at therapeuticdosages which are typically in milligram/g.

4. Conclusion

In summary, the proposed method is fast and suitable for theanalysis of 11 weight loss drugs in dietary supplements. Thismethod is validated for LC-MS/MS amendable drug substancesin dietary supplements. This method extends the capability ofthe FDAs dietary supplement program to identify and quantifyof adulterated weight loss drugs in dietary supplements usingLC-MS/MS.

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5. Declaration of Conflicting Interest

The authors declare that there is no conflict of interest. Re-search was funded by U.S. Food and Drug Administration.

6. Disclaimer

The views expressed are those of the authors and should notbe construed to represent the views or policies of the U.S. Foodand Drug Administration. Any reference to a specific commer-cial product, manufacturer, or otherwise, is for the informationand convenience of the public and does not constitute an en-dorsement, recommendation or favoring by the U.S. Food andDrug Administration.

7. Acknowledgment

We would like to thank Mrs. Annie Rice for reading themanuscript and comments.

8. Article information

The article was received on October the 15th, 2015 andavailable on-line January the 7th, 2016.

References

[1] P. R. F. Song, D. Monroe, A. Demerdash, C. Palmer, Screening for mul-tiple weight loss drugs in dietary supplement materials by flow injectiontandem mass spectrometry and their confirmation by liquid chromatogra-phy tandem mass spectrometry, Laboratory Information Bulletin 4549.

[2] F. M. S. R. Gratz, C. L. Flurer, K. A. Wolnik, Analysis of undeclaredsynthetic phosphodiesterase-5 inhibitors in dietary supplements and herbalmatrices by lc-esi-ms and lc-uv, J. Pharm. Biomed. Anal. 36.

[3] I. . S. E. Edison, S. R. Gratz, B. M. Gamble, A. S. Mohrhaus, Lc-ms/msmethod for sibutramine and related compounds, Laboratory InformationBulletin 4477.

[4] Tainted products marketed as dietary supplements potentially danger-ous. www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/2010/

ucm236967.htm Web accessed December 2015.[5] Dietary Supplement Analysis Quality Assurance Program.

http://www.nist.gov/mml/csd/organic/dsqap.cfm. Web accessed December2015.

[6] Guidance for Industry Mass Spectrometry for Con-firmation of the Identity of Animal Drug Residues.http://www.fda.gov/downloads/AnimalVeterinary/ GuidanceCompli-anceEnforcement/GuidanceforIndustry/ucm052658.pdf. Web accessedDecember 2015.

[7] Slim-30 Herb Supplement: Undeclared Drug Ingredi-ent. http://www.fda.gov/Safety/MedWatch/ SafetyInforma-tion/SafetyAlertsforHumanMedicalProducts/ucm219463.htm. Webaccessed December 2015.

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journal of regulatory science 02 (2015) 15–19 method

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